The determination of the three dimensional atomic structure of matter is one of the most important areas of pure and applied research. This field, known as X-ray crystallography, utilizes the diffraction of X-rays from crystals in order to determine the precise arrangement of atoms within the crystal. The result may reveal the atomic structure of substances as varied as metal alloys to the structure of deoxyribonucleic acid (DNA). Some of the greatest discoveries in the history of science have been made by crystallographers. The limiting step in all of these areas of research involves the growth of a suitable crystalline sample.
One important and rapidly growing field of crystallography is protein crystallography. Proteins are polymers of amino acids and contain thousands of atoms in each molecule. Considering that there are 20 essential amino acids in nature, one can see that there exist virtually an inexhaustable number of combinations of amino acids to form protein molecules. Inherent in the amino acid sequence or primary structure is the information necessary to predict the three dimensional structure. Unfortunately, science has not yet progressed to the level where this information can be obtained apriori. Although considerable advances are being made in the area of high field nuclear magnetic resonance, at the present time, the only method capable of producing a highly accurate three dimensional structure of a protein is by the application of X-ray crystallography. This requires the growth of reasonably ordered protein crystals (crystals which diffract X-rays to at least 3.0 angstroms resolution or less).
Because of the complexity of proteins, obtaining suitable crystals can be quite difficult. Typically several hundred to several thousand individual experiments must be performed to determine crystallization conditions, each examining a matrix of pH, buffer type, precipitant type, protein concentration, temperature, etc. This process is extremely time consuming and labor intensive. In this regard, the field is often considered more of an art than a science and skilled practitioners are highly valued. The resulting three dimensional structure produced from the protein crystals can have enormous implications in the fundamental understanding of molecular biology such as how enzymes perform various catalytic activities, switch on biological pathways, or transport molecules within the circulatory system. In the past few years the determination of protein structures important as therapeutic targets has made possible the rational design of new more effective pharmaceuticals.
Recent advances in this field such as high speed computer graphics and X-ray area detection technologies has revolutionized the pace at which the three-dimensional structures can be determined. Still, however, the bottle neck has been the determination of conditions necessary to grow high quality protein crystals. In order for protein crystals to be suitable for structural analysis via X-ray diffraction methods, crystals on the order of about 0.5 mm in diameter or greater must be obtained depending on the intrinsic quality of the protein crystal, the size of the unit cell, and the flux of the X-ray source, etc. This has proved extremely inconvenient and difficult to accomplish on a consistent basis using techniques and crystallization trays known at present.
At present, proteins and other small molecules are crystallized by a variety of conventional experimental methods. Among these many methods, there are three that are most commonly used in the art. One of the main techniques available for growing crystals, known as the hanging-drop or vapor diffusion method, is a method wherein a drop of a solution containing protein is applied to a glass cover slip and placed upside down in an apparatus such as a vapor diffusion chamber where conditions lead to supersaturation in the protein drop and the initiation of precipitation of the protein crystal. However, this method is usually troublesome and inefficient because current methods of employing this technique to achieve crystal growth are somewhat primitive, whether conducted manually or through robotic devices, and involve a series of adjustments of the conditions until a suitable experimental regimen is found. In typical screening methods under this process, it is generally required that the lab technician vary the conditions of pH, buffer type, temperature, protein concentration, precipitant type, precipitant concentration, etc., for each set of experiments, and even adjusting for the myriad of conditions, often only minute samples of the protein can be studied at one time. These variables create an extensive and complex matrix of small experiments, with each series requiring another set of protein drops to be affixed to the glass cover slips and inverted and sealed in the vapor pressure chamber. As presently carried out using currently available devices, crystal growth methods such as the hanging drop method are tedious, time-consuming, and hard to carry out successfully and efficiently with reproducibility.
In another method referred to as the dialysis method, the protein solution is contained within a semipermeable size exclusion membrane and then placed in a solution of fixed pH, precipitant concentration, etc., as in the reservoir solutions prepared for the hanging-drop method. As the precipitant diffuses through the membrane into the protein compartment, the solubility of the protein is reduced and crystals may form. Both vapor diffusion and dialysis methods require extensive screening of numerous variables to achieve the desired results.
Unfortunately, it has been observed that crystal growth carried out under normal gravitational conditions suffer from turbulent convective flows which occur in the above described methods. In particular, during crystal growth under 1 g, the solute depleted regions surrounding a growing crystal normally produce these turbulent convective flows which appear to have significant effects on the crystal quality. For methods such as liquid--liquid diffusion and dialysis, which require the diffusive mixing of two solutions of greatly differing densities, the elimination or reduction of these density driven convective flows is of the utmost importance if one is to successfully carry out crystal growth.
Still another method of protein crystal growth involves what is referred to as gel crystal growth. This method involves the placement of a gel into the end of small diameter glass capillaries. After the solutions have gelled, a protein solution is placed into one end (top) of the capillary and the other end is submerged in a solution of precipitating agent. If the conditions are appropriately selected, crystal growth occurs at a point in the gel where the protein and precipitating agent reach the proper concentrations as the solutions slowly mix by diffusion. Since this is a diffusion limited process, it thus only occurs after an extended period of time. Crystals however, grown by this method are often larger and of higher quality. The approach to screening for the proper crystallization conditions entails the use of numerous bottles of precipitant solutions containing glass capillaries. The method is thus cumbersome and has the disadvantage of that once the crystals are formed in the gels it is extremely difficult to remove them without damage.
In short, the currently accepted practice of screening for protein crystallization conditions suffers from a myriad of problems which have limited the use of high resolution x-ray crystallographic methods in the determination of the three dimensional structures of the protein molecules. It is thus highly desirable in light of the recent advances in the field of protein crystallography to develop highly efficient, simple, and effective methodologies for obtaining the desired conditions for the growth of high quality protein crystals for x-ray crystallography, and yet which can also avoid the problems associated with the prior art devices, and in this respect, the present invention addresses this need and interest.